There has been a growing interest in enriching rare cells (e.g., for subsequent isolation and characterization) over the past several years. This may be attributed at least in part to the recognition that rare cells, such as cancer cells, can provide information that is helpful in the diagnosis and/or treatment of various medical conditions.
The desire to enrich cancer cells is based in part on the knowledge that a majority of cancer deaths occur due to the metastasis of tumors. As such, the presence of carcinoma cells in the peripheral blood is an indication of cancer cell spread, and enriching such cancer cells would be of great diagnostic benefit. This need is particularly acute in prostate cancer, wherein approximately two-thirds of such cancers are clinically localized at the time of diagnosis, but only about half of these prove to be confined to the prostate at the time of surgery. Thus, nearly one-third to one-half of cancers have spread beyond the prostate when first identified, cancers which could be detected at any earlier stage if accurate, highly sensitive enrichment methods were available.
Much of the activity with respect to the early detection of prostate cancer has centered around the usefulness of serum prostate specific antigen (PSA). However, PSA is organ specific and not cancer specific, and is produced by normal, benign, and malignant prostate epithelium. As a result, the positive predictive value for PSA as a screen for prostate cancer is generally less than 50 percent.
Additionally, the maximal level of cancer cells in the peripheral blood has been estimated to be two in 10.sup.7 leukocytes. Fidler, Cancer Res., 50, 6130 (1990). Thus, while studies have suggested that prostate cancer cells circulate in the bloodstream of men with advanced disease, it is difficult to detect these few circulating cancer cells.
Methods for separating and detecting cancer cells have included, for example, using immunomagnetic beads and the polymerase chain reaction (e.g., Hardingham et al. Cancer Res., 53, 3455 (1993)), using density gradient gels (e.g., U.S. Pat. No. 4,255,256), or using density gradient centrifugation followed by immunological separation to bind the cancer cells (e.g., Griwatz et al., J. Immunol. Methods., 183, 251-265 (1995)).
These methods have been generally unsatisfactory as they lack the efficiency and sensitivity to separate the few cancer cells in a blood sample. Additionally, these methods may provide low cell recovery, since the highly fragile cancer cells can be damaged during the separation process and/or the relatively sticky cancer cells can become inappropriately bound during the separation process.
For example, conventional processes utilize "positive selection", wherein a rare cell is bound to a binding agent such as an antibody, and the bound rare cell is separated from the non-rare cells. Thereafter, the rare cell is separated from the antibody by heat or other suitable means, which can damage or destroy the rare cell, making it difficult to detect and/or culture. Additionally, or alternatively, some processes involve concentrating cancer cells by centrifugation. However, since some cancer cells are fragile and/or tend to stick to surfaces onto which they come into contact, these processes can also damage or destroy the rare cells, which is undesirable as described above. Furthermore, some processes provide for "fixing" the cells during the separation process, thus rendering them unsuitable for culturing or PCR analysis.
In view of the foregoing, there exists a need for an efficient, highly sensitive and highly reproducible method for enriching rare cells from a population of cells. There is also a need for a method that can minimize damage to those rare cells that are fragile and/or sticky.
The present invention provides for ameliorating at least some of the disadvantages of the prior art. These and other advantages of the present invention will be apparent from the description as set forth below.